Received: 14 May 2018 Revised: 22 December 2018 Accepted: 27 December 2018 DOI: 10.1111/pce.13516

ORIGINAL ARTICLE

Anatomical changes with needle length are correlated with structural and physiological traits across five Pinus species

Na Wang1,2 | Sari Palmroth2 | Christopher A. Maier3 | Jean‐Christophe Domec2,4 | Ram Oren2,5

1 School of Forestry, Northeast Forestry University, Harbin 150040, China Abstract 2 Nicholas School of the Environment, Duke The genus Pinus has wide geographical range and includes species that are the most University, Durham, NC 27708, USA economically valued among forest worldwide. Pine needle length varies greatly 3 Southern Research Station, USDA Forest among species, but the effects of needle length on anatomy, function, and coordina- Service, Durham, NC 27709, USA 4 Bordeaux Sciences Agro, UMR 1391 INRA- tion and trade‐offs among traits are poorly understood. We examined variation in leaf ISPA, 33175 Gradignan Cedex, France morphological, anatomical, mechanical, chemical, and physiological characteristics 5 Department of Forest Sciences, University of among five southern pine species: Pinus echinata, Pinus elliottii, Pinus palustris, Pinus Helsinki, Helsinki, Finland Correspondence taeda, and Pinus virginiana. We found that increasing needle length contributed to a Sari Palmroth, Nicholas School of the trade‐off between the relative fractions of support versus photosynthetic tissue Environment, Duke University, Durham, NC 27708, USA. (mesophyll) across species. From the shortest (7 cm) to the longest (36 cm) needles, Email: [email protected] mechanical tissue fraction increased by 50%, whereas needle dry density decreased Na Wang, School of Forestry, Northeast by 21%, revealing multiple adjustments to a greater need for mechanical support in Forestry University, Harbin 150040, China. Email: [email protected] longer needles. We also found a fourfold increase in leaf hydraulic conductance over Funding information the range of needle length across species, associated with weaker upward trends in National Science Foundation, Grant/Award stomatal conductance and photosynthetic capacity. Our results suggest that the leaf Number: NSF‐IOS‐1754893; Erkko Visiting Professor Programme of the Jane and Aatos size strongly influences their anatomical traits, which, in turn, are reflected in leaf Erkko 375th Anniversary Fund, through the mechanical support and physiological capacity. University of Helsinki

KEYWORDS

conductance, leaf structure, leaf traits, nitrogen, photosynthesis, pine, hydraulics, stomata, water relations, xylem transport

1 | INTRODUCTION the leaf boundary‐layer conductance and thus water loss rate and leaf temperature (Yates, Verboom, Rebelo, & Cramer, 2010). Furthermore, Leaf size exhibits great plasticity, both along environmental gradients leaf size affects plant productivity and adaptive capacity to changes in and within communities, varying by over 100,000‐fold among species its environment (Pickup, Westoby, & Basden, 2005; Westoby, Falster, worldwide (Milla & Reich, 2007; Wright et al., 2017). Corner's Moles, Vesk, & Wright, 2002; Wright, Falster, Pickup, & Westoby, hypothesis states that, compared with species with smaller , 2006). A better understanding of constraints on the structural design species with large leaves have greater proportions of their shoot bio- of leaves, and the resulting coordination or trade‐offs among leaf traits mass invested in foliage and hold more widely spaced leaves on shoots across species, is key to understanding variation of plant growth rates (Corner, 1949). Thus, species with large leaves tend to experience less and adaptive and competitive capabilities. self‐shading within a shoot (Falster & Westoby, 2003). Leaf size The genus Pinus has a wide geographical range and includes affects, or is related to, many functional traits. Combined with leaf species that are the most economically valued among forest trees organization on shoots, leaf size affects light interception efficiency worldwide (Wear & Greis, 2002). Among , pines species show and the net carbon assimilation capacity of a plant, in part by affecting the largest range of needle length, yet relatively little is known about

1690 © 2019 John Wiley & Sons Ltd wileyonlinelibrary.com/journal/pce Plant Cell Environ. 2019;42:1690–1704. WANG ET AL. 1691 the influence of needle length on needle structure and function and needle length to irradiance on needle surfaces. That is, in a given light how variations in anatomical traits affect mechanical and physiological environment, mutual shading among needles of a shoot (inversely traits. More is known about how needle length and organization affect related to STAR) STAR appears to reach a minimum at intermediate light distribution on needle surfaces (Niinemets, Tobias, Cescatti, & needle lengths, providing the highest mean irradiance on needle Sparrow, 2006; Stenberg, Palmroth, Bond, Sprugel, & Smolander, area relative to shoots with either shorter or longer needles. If light 2001; Thérézien, Palmroth, Brady, & Oren, 2007). The amount of light availability was the main driver of photosynthetic capacity and related intercepted by unit of leaf area depends on its location relative to leaf traits, these traits should show a similar pattern with needle length other shading elements in the canopy. In coniferous canopies, three‐ among species. dimensional foliage elements are grouped into shoots, that is, (semi‐) However, mechanical traits control the physical response of annual growth units, often considered the basic functional units. The leaves to environmental forces, with physiological and ecological con- mean light intensity, or mean irradiance, on needle surfaces of a shoot sequences (Niklas, 1999). Needles work as cantilevered mechanical depends on the amount of light reaching the shoot and on the shoot “devices,” bearing a relatively uniform load along their length. They interception efficiency. This efficiency varies with the size and orienta- must be sufficiently rigid to support their own weight mechanically tion of the shoot and the dimensions and organization of the needles against the pull of gravity yet flexible enough to bend or twist without (Niinemets, Tobias, et al., 2006; Stenberg et al., 1994). Ceteris paribus, breaking when subjected to strong and dynamic external forces, such the mean shoot silhouette‐to‐total leaf area ratio ( STAR; Stenberg as wind, snow, hail or ice formation, animal action, abrasion against et al., 1994) first increases with needle length, reaching a maximum other canopy elements, and falling debris (Raupach & Thom, 1981; and decreasing thereafter with further increases in length (Figure 1a, Vogel, 1989). Previous studies demonstrated that the investment in simulated using the model by Thérézien et al., 2007). This simplified support biomass (midrib and petiole mass) per unit leaf area (or leaf analysis does not account for other shoot structural characteristics, fresh mass) in larger leaves is greater than in smaller leaves (Niinemets, which vary among species, yet suggests a hypothesis that directly links Portsmuth, & Tobias, 2006). In conifers, the bending force operating on a needle is proportional to the cube of its length (Niklas, 1992, 1999); thus, the biomass or volume fraction of supporting tissues (epidemical tissue plus xylem) must scale exponentially with needle length to main- tain a given needle angle. However, among pine species, needle cross section does not increase with increasing needle length (Figure 1b, Table S1). This suggests that the extra structural support required for longer needles is not achieved simply through increased cross‐ sectional area and associated strength but by increasing the relative investment in mechanical tissues. The hydraulic costs and benefits of deploying a given leaf area as many small versus fewer large leaves are still a matter of debate (Corner, 1949; Givnish, 1984; Niinemets, Portsmuth, & Tobias, 2006; Yang, Li, & Sun, 2008). The pathway of water within leaves represents a substantial portion of the whole‐plant resistance to water flow from root to the substomatal air space (Nardini & Salleo, 2000; Sack & Holbrook, 2006). Thus, for example, correlations have been noted

among leaf hydraulic conductance (Kleaf), maximum stomatal conduc-

tance (maximum gs), transpiration, and photosynthetic rates across species (Brodribb, Feild, & Jordan, 2007; Sack, Cowan, Jaikumar, & Holbrook, 2003; Sack & Holbrook, 2006), with additional variation caused by resource availability (Domec et al., 2009; Sellin, Sack,

Õunapuu, & Karusion, 2011). In angiosperms, Kleaf varies indepen- dently of leaf size among species because of the overwhelming effects of the great phylogenetic and developmental diversity of the vascular architecture (Sack et al., 2003; Sack & Frole, 2006). In contrast to the complex leaf vein patterns in angiosperm, pine needles have a very simple design that consists of a single middle vein (Esau, 1977; Zwieniecki, Stone, Leigh, Boyce, & Holbrook, 2006). However, whether the diversity in needle length translates to large differences in water transport capacity and, thus, in stomatal conductance and FIGURE 1 Variation in modelled spherically averaged shoot‐ gas exchange remains unclear. silhouette‐to‐total‐leaf‐area ratio (STAR; a), and needle cross‐ The hydraulic conductance of xylem, and along the flow paths sectional area (b) with needle length. The model used in (a) is from Thérézien et al. (2007). The data in (b) were taken from published outside the xylem to evaporation sites, largely depends on the dimen- papers listed in Table S1 sions and anatomy of the leaf (Brodribb et al., 2007; Scoffoni et al., 1692 WANG ET AL.

2016). Unknown at this time, however, is whether variation among would decrease with needle length (H1b). Driven by the increase species in needle length is accompanied by variation in the anatomical in leaf hydraulic conductance, stomatal conductance and maximum properties of the xylem and of tissues outside the xylem (transfusion photosynthetic rates would also increase with needle length and mesophyll) and how these variations translate to differences in (H1c). However, if the variation of light availability on needle surfaces, leaf function. These differences could manifest in variation of induced by shoot structure through varying needle length (Figure 1a), traits such as the hydraulic conductance of outside‐xylem pathway was the main driver of photosynthetic capacity and related leaf traits,

(Koutside‐xylem), xylem (Kxylem), or their composite, Kleaf, which repre- the relationship between leaf gas exchange characteristics and needle sents the whole‐leaf water transport capacity. For example, although length would be concave as the amount of light on needle surfaces needle cross‐sectional area remains relatively constant with needle (relative to available light) declines at the higher portion of the range length (Figure 1b), the volume fraction of mechanical tissues could in needle length (H2). increase with length, forcing a reduction in the fraction of mesophyll tissue, along with the path length within the mesophyll, potentially 2 | MATERIALS AND METHODS decreasing the hydraulic resistance along the radial outside‐xylem pathway. This could increase Koutside‐xylem and thus Kleaf with needle length (Brodribb et al., 2007; Domec, Palmroth, & Oren, 2016; 2.1 | Site description Scoffoni et al., 2016) and the photosynthetic rate, if it is conductance ‐ ‐ limited. However, if it is limited by the amount of photosynthetic In 2011, 1 year old seedlings of P. echinata Mill., P. elliottii Engelm., machinery per unit of area (e.g., the ratio of mesophyll cell surface area P. palustris Mill., P. taeda L. (two families), and P. virginiana Mill. were to leaf surface area; Nobel, Zaragoza, & Smith, 1975) and/or by meso- planted in 32 m × 40 m plots, with 4 m × 2 m spacing, on a site with phyll diffusion conductance (Kuusk, Niinemets, & Valladares, 2018b), sandy loam soil of the Appling series in the Duke Forest, Durham, ′ ′ such adjustments may reduce photosynthetic rates. NC (36°01 N, 78°59 W). P. echinata and P. elliottii plots were planted ‐ With increasing (average) light availability, the amount of photo- with the second generation seedlings from Flint River Nursery, Byromville, GA. P. palustris seedlings are of various provenances synthetic machinery and biochemical demand for CO2 (photosynthetic ‐ capacity) increase, along with the evaporative force driving transpira- across the south eastern United States (Longleaf Pine Regional Provenance/Progeny Trial, NC State University Cooperative Tree tion, and must be met by an enhanced supply of CO2 and liquid water Improvement Program and USDA Forest Service, Raleigh, NC). P. taeda through higher Kleaf and gs (Brodribb et al., 2007; Scoffoni et al., 2016). ‐ Thus, needle length may affect both the mean irradiance on needle seedlings are from two mass control pollinated families (ArborGen, ‐ ‐ surfaces, as well as the hydraulic capacity for liquid water delivery Inc., Ridgeville, SC), with broad (AGM 37) and narrow (AGM 22) required to keep stomata open, facilitating the use of captured solar crown characteristics, from Supertree Nursery, Blenheim SC. energy in photosynthesis. This study aims at assessing the combined P. virginiana seedlings were from Claridge Nursery, Goldsboro, NC. effect of needle length on variables that affect both leaf display to At the end of the 2016 growing season, the dominant heights of the light and hydraulic capacity and the consequences to stomatal trees were as follows: P. taeda, 6.6 m; P. elliottii, 4.4 m; P. virginiana, conductance and photosynthetic rate. 3.9 m; and P. echinata, 3.3 m. P. palustris trees showed a large range We studied the five most common pine species native to the of heights due to the distinctive developmental pattern of the species south‐eastern United States, namely, Pinus echinata, Pinus elliottii, (averaging 1.4 m in our plot), but the individuals sampled had passed “ ” ‐ Pinus palustris, Pinus taeda (two families), and Pinus virginiana, the grass and bottlebrush stages and were ~2 m tall in the fall of exhibiting a wide range in needle length. Except for P. virginiana,a 2015. The climate of the region is warm and humid with mean annual member of subsection Contortae, the others are called “southern temperature of 15.5°C. Annual precipitation is 1,145 mm and pines” (subsection Australes). All five species are shade intolerant, distributed evenly throughout the year. and among them, P. palustris is the least, and P. virginiana is the most shade tolerant. All five species are relatively well adapted to poor soils 2.2 | Hydraulic measurements and show overlapping geographical ranges, each with at least two others. The evolutionary relationships of the southern pines remain Hydraulic measurements were conducted in October 2015 on unresolved (Koralewski, Mateos, & Krutovsky, 2016), and in this study, branches collected from the upper crowns of five randomly sampled we assess leaf trait values of these co‐occurring and related species as individuals per species (n = 5). To prevent pine resin from causing independent solutions to match energy capture by leaf surfaces with extraneous surface resistance through resin canal breakages, we water supply to stomata. We aimed at exploring the potential mecha- soaked the end surfaces in water for 60 to 90 min before the first nisms by which needle length affects leaf gas exchange in response to measurements to remove excess resin and then carefully recut the structural and hydraulic adjustments. We examined the variation in end‐surfaces with a clean razor blade (Booker, 1977; Melcher et al., anatomical, mechanical, chemical, hydraulic, and gas exchange traits 2012). We describe below hydraulic resistance (R) rather than hydrau- with needle length among the five species, growing under the same lic conductance (K, e.g., Rleaf rather than Kleaf =1/Rleaf), because resis- climatic and edaphic forcing. We hypothesize that the volume fraction tances add in series, enabling measured values to be partitioned into of mechanical tissue (epidermal tissue plus xylem) would scale their resistance components. The methodology used in this study is with needle length (H1a), whereas the fraction of mesophyll tissue described in detail elsewhere (Domec et al., 2016). Briefly, leaf hydrau- and, consequently, the outside‐xylem and leaf hydraulic resistances lic resistance (Rleaf) was measured by a high‐pressure flow metre WANG ET AL. 1693

(HPFM; Tsuda & Tyree, 2000). Apical branches were connected to the mesophyll cells (Smesophyll cell, Cmesophyll cell) were also recorded. Images HPFM (Dynamax Gen2, IN) with a water tight seal. Deionized, taken at 400× were used to determine the xylem and phloem areas and degassed water, filtered at 0.1 μm, was forced at a pressure of 0.2– the number and size of tracheids within the xylem area. From these

0.3 MPa into the shoots through the stem and eventually out through measurements, mesophyll cells surface area (Smesophyll‐surf.) per the stomata. Temperature was automatically recorded by the HPFM, needle surface area (Sneedle) was calculated as Smesophyll‐surf./ and all measurements were corrected to values at 25°C. Branch Sneedle =(Smesophyll/Smesophyll cell)×Cmesophyll cell/C. Xylem hydraulic ∑n 5 ∑n 4 hydraulic resistance (Rbranch) was shown to increase during the first diameter was calculated as i¼1di / i¼1di , where d is the diameter 3–5 min, and data points were recorded every 30 s after the flow rate of the ith tracheid and n is the number of the tracheids in the xylem became stable. After Rbranch was measured, the needles were removed (Kolb & Sperry, 1999). In our study, mechanical (or supporting) tissue with a fresh razor blade at their connections to the woody stem and was defined as epidermal tissues plus xylem; therefore, mechanical the hydraulic resistance reassessed (Rstem). Because shoot resistances tissue fraction (%) was calculated as the ratio of the sum of epidermal are added in series, Rleaf was calculated as Rleaf = Rbranch − Rstem. tissues area and xylem area to needle cross‐sectional area. Hydraulic resistance was measured on both control (intact) leaves To estimate dry/fresh mass ratio, dry (and fresh) density of and on frozen and then thawed needles. Freezing the needles removes needles, leaf mass per unit area, and leaf mass per unit length, three most of the hydraulic resistances of the outside‐xylem water pathway needle samples (made of 20–30 needle fascicles each) per tree from (Cochard, Froux, Mayr, & Coutand, 2004; Domec et al., 2016; Nardini, five trees per species were collected. Dry/fresh mass ratio was calcu-

Salleo, & Raimondo, 2003; Tyree, Nardini, & Salleo, 2001), and Rleaf lated as the ratio of needle dry mass to needle fresh mass. Dry and estimated from such needles represents that of the leaf xylem water fresh density was calculated as the ratio of needle dry and fresh mass, pathway (Rxylem). To estimate Rxylem, which represents the inverse of respectively, to needle fresh volume. Leaf mass per unit area was the axial leaf xylem conductance, we measured Rbranch and Rleaf on calculated as the ratio of needle dry mass to total needle surface area – − −2 frozen thawed branches (frozen at 55°C for 10 min, and then (LMAtotal‐surf.,gm ). Leaf mass per unit length was calculated as the thawed for 30 min at +23°C). Preliminary tests on four shoots showed ratio of needle dry mass to total needle length (g m−1). Fresh mass that, in fact, 15 min was sufficient to completely thaw the samples of needles was obtained, followed by fresh needle volume based on because the branches were plunged in water at room temperature water displacement method. After drying the surface of the needles before connecting to the HPFM and that Rbranch, once stable, with a paper towel, needles were scanned. Projected area and needle remained so for >20 min, the period during which removing needles length were estimated by analysing the images with the software and remeasuring were completed (Domec et al., 2016). It should also Image‐J (National Institutes of Health, MD). Total surface area was be noted that, in our experiments, the freeze–thaw process did not calculated as projected area × π following empirical cause embolism because thawing did not occur under tension studies demonstrating that the ratio is not significantly different from (Pittermann & Sperry, 2006). Hydraulic resistance of the pathway that quantity (Johnson, 1984; 3.08 ± 0.21 SD for six pine species, outside the leaf xylem, the extravascular leaf resistance, was calcu- including P. taeda and P. virginiana; Oker‐Blom & Smolander, 1988; lated as Routside‐xylem = Rleaf − Rxylem. Finally, we converted resistance 3.00 ± 0.28; Oker‐Blom, Kaufmann, & Ryan, 1991; 3.09 ± 0.33). to conductance, normalizing all hydraulic conductance values by the Finally, needles were oven dried at 65°C for 48 hr to a constant mass total needle surface area of the branch. and weighed again.

2.3 | Leaf structure and anatomy 2.4 | Gas exchange measurements

In March 2017, needles were sampled from randomly assigned trees Gas exchange was measured during two campaigns in 2017. On April (five needles per tree and five trees per species) for structural and 26th–27th, light‐saturated photosynthetic rates (A) and correspond- anatomical measurements. Samples were placed in plastic bags, ing stomatal conductance (gs)of1‐year‐old needles on intact transported to laboratory, and kept in a cold room until processing upper‐crown branches were measured using three Li‐Cor 6400 gas (within hours from collection). Free‐hand transverse sections (with exchange system (with 6400‐02B red/blue light source, and thickness of ~40 μm) of fresh needles were cut in the midsections of 20 × 30 mm chamber; Li‐Cor Biosciences, Lincoln, NE). Ten trees the samples using fresh razor blades. Sections thicker than 100 μm (“gas exchange trees”) of each species were randomly selected, approach mesophyll cell size and may lead to erroneous estimates of and one set of measurements was taken from each tree (n = 10). some measures. The sections were photographed at 40×, 100×, and Midsections of two to three sun‐acclimated fascicles were enclosed 400× magnification by a digital camera mounted on a microscope, in the chamber. After an acclimation period of a few minutes, until and the images were analysed using the Motic Images Advanced 3.2 steady gas exchange rates were reached, we recorded the readings software (Motic Corporation, Zhejiang, China). Images taken at 40× at 30‐s intervals for 5 min. Chamber conditions were maintained were used to determine cross‐sectional width, thickness, circumfer- close to ambient conditions. That is, (block) temperature were set to ence (C), needle cross‐sectional area, epidermal tissues area, and meso- near ambient temperature (around 28°C during these 2 days) and −2 −1 phyll area (Smesophyll). Images taken at 100× were used to determine quantum flux density to 1,800 μmol m s . We did not control the area of central cylinder, vascular bundle, and transfusion tissue. water vapour pressure in the chamber, and the relative humidity Thickness of mesophyll tissue and area and circumference of individual ranged from 50% to 70%. 1694 WANG ET AL.

On June 2nd–4th and 8th–10th, photosynthetic responses to max were expressed both on total area basis (Aarea, Vc, max‐area) and variations in leaf internal CO2 concentration, that is, A–Ci curves, were on mass basis (Amass, Vc, max‐mass), which were calculated as Aarea/ measured. Curves were developed on 1‐year‐old foliage of detached LMAtotal‐surf. and Vc, max‐area/LMAtotal‐surf. Stomatal conductance was shoots collected from five of the ten gas exchange trees (n = 5). The expressed on total needle surface area basis. cut branches were recut under water and kept well hydrated during the measurements. For each sample, a single curve over 11 chamber 2.5 | Statistical analysis CO2 concentrations (400, 50, 100, 150, 200, 300, 400, 800, 1,000, 1,200, and 1,500 ppm) was measured. Other environmental variables Individual trees were used as replicates. The effect of species on leaf in the chamber were maintained as described above. Maximum rates anatomical, mechanical, chemical, and hydraulic traits were tested of carboxylation (V ) were determined at a standard temperature c, max using one‐way analysis of variance and the effect of species on gs of 25°C for each needle sample with the Farquhar biochemical model was tested by analysis of covariance incorporating the natural loga- of photosynthesis (Farquhar, von Caemmerer, & Berry, 1980) as rithm of leaf‐to‐air water vapour pressure deficit (VPD) as a covariate. described by Medlyn et al. (2002) and the Rubisco kinetic characteris- Fisher's least significant difference test was used to identify the differ- tics obtained from Bernacchi, Singsaas, Pimentel, Portis, and Long ences in those leaf traits among tree species. Linear regressions were (2001). We did not correct gas exchange rates for chamber diffusion used to evaluate relationships between leaf functional traits and nee- leaks (Rodeghiero, Niinemets, & Cescatti, 2007). However, all our dle length, and among leaf traits, across species. Probability levels measurements were carried out with settings that minimize the leaks, P < 0.05 and P < 0.10 were considered to indicate a significant rela- that is, in humid field conditions, and with chamber conditions set tionship and a tendency, respectively. – close to ambient conditions (excluding [CO2] for A Ci curves). More- Leaf hydraulic traits and gas exchange were measured on separate over, because the cross‐sectional dimensions were fairly similar among campaigns, in 2015 and 2017, respectively. The laboratory measure- the species in our study (Table 1), we assumed that the effect of any ments of leaf hydraulics represent optimal conditions at full saturation unavoidable leaks on V and J estimates were also similar c, max max (and thus maximum Kleaf), and therefore, the means of gs and Aarea by across species, facilitating valid comparisons. species estimated in April (and under close to saturated soil moisture After each set of gas exchange measurements, needle length and conditions) were used to analyse their potential relationships with width were measured, and the central angle of the needles was calcu- the respective species‐specific Kleaf. Because the measured gs lated (2π/# needles per fascicle) to estimate needle surface area in the appeared sensitive to changes of VPD, the mean gs and Aarea of all chamber by assuming that needle cross section represents a circle sec- the species were normalized to VPD at 1.5 KPa, which represents tor. To test the approach, we measured both needle width and needle the mean VPD over all the measurements. Statistical analyses were circumference from cross sections (four needles per tree and five trees performed using the SPSS software (version 19.0, SPSS, Inc., Cary, per species) and calculated the ratio of circle sector‐based estimate NC), and curve fits were performed using SigmaPlot (version 12.5, circumference (Ccircle sector) to measured circumference (C). For com- Systat Software, Inc., San Rafael, CA). parison, we also calculated circumference assuming a pie‐shaped cross section (Cpie; Niinemets, Ellsworth, Lukjanova, & Tobias, 2002) and the 3 | RESULTS ratio of Cpie to C. The sampled fascicles were then oven dried to a con- stant mass at 65°C (for 48 hr), weighed and ground to determine foliage nitrogen (N) concentration with a Carlo‐Erba analyser (Model 3.1 | Variation in anatomical and morphological NA 1500, Fison Instruments, Danvers, MA; USDA Forest Service Lab- characteristics with needle length oratory, Research Triangle Park, NC). Nitrogen concentration was −2 expressed on needle total surface area basis (Narea,gm ), on meso- Across species, needle length ranged from 7.5 ± 0.3 cm in P. virginiana −2 phyll cells surface area basis (Nmesophyll‐surf.,gm , calculated as to 34.6 ± 1.3 cm in P. palustris. In needle cross sections, the phloem

Narea/(Smesophyll‐surf./Sneedle)), and on mass basis (Nmass, %). A and Vc, area remained constant at ~2.1% over the range of needle lengths

TABLE 1 Needle morphological characteristics in five Pinus species (mean ± SE)

Pinus taeda Needle morphological Pinus virginiana Pinus echinata Pinus elliottii Pinus palustris characteristics (PV) (PEC) (PT2) (PT1) (PEL) (PP) P value Needle length (cm) 7.51 ± 0.35d 9.28 ± 0.57d 17.32 ± 0.52c 19.69 ± 0.41c 23.23 ± 1.37b 34.59 ± 1.31a 0.0001 Width (mm) 1.69 ± 0.03a 1.48 ± 0.02be 1.41 ± 0.04de 1.50 ± 0.04be 1.58 ± 0.03bc 1.61 ± 0.03ac 0.0001 Thickness (mm) 0.78 ± 0.03a 0.74 ± 0.02a 0.62 ± 0.02b 0.63 ± 0.02b 0.76 ± 0.02a 0.80 ± 0.02a 0.0001 Cross‐sectional area (mm2) 1.01 ± 0.05a 0.76 ± 0.05b 0.59 ± 0.03d 0.64 ± 0.03d 0.90 ± 0.04ac 0.86 ± 0.03bc 0.0001 Circumference (mm) 4.15 ± 0.08a 3.55 ± 0.08cd 3.27 ± 0.09ef 3.44 ± 0.08cf 3.89 ± 0.09b 3.76 ± 0.07bd 0.0001

−2 a b b BC ac bc LMAtotal‐surf. (g m ) 98.87 ± 2.50 80.78 ± 4.11 81.49 ± 5.14 83.52 ± 1.60 91.96 ± 3.75 82.94 ± 1.64 0.005 Dry/fresh mass (%) 37.57 ± 0.69a 32.58 ± 0.77d 36.72 ± 0.38ac 37.05 ± 0.29a 38.39 ± 0.64a 35.22 ± 0.63bc 0.0001

Note. Different lower case letters indicate significant differences (P < 0.05) in needle morphological characteristics among species according to Fisher's least significant difference test. LMA: leaf mass per area. WANG ET AL. 1695

(Figures 2 and S1a), and the fractions of xylem, vascular bundle, trans- diameter increased ~27% across the range in needle length (Figure 4 fusion tissue, central cylinder, and epidermal tissue increased with d). Taken together, the longer the needle, the larger was the fraction needle length (Figures 2 and S1b–f). Although the xylem comprised a of its cross‐sectional area made of xylem tissue containing larger tra- small fraction (2.2 ± 0.1% to 4.5 ± 0.2%) of the cross‐sectional area, cheids. The increase in the supporting tissue (seen Figure 4b) is attrib- it showed the largest relative increase of 108% with needle length, utable to the increasing fraction of both the epidermal tissue and the followed by the vascular bundle area, increasing 71%, from xylem (Figure 5a). Furthermore, the fraction of mesophyll tissue was 3.9 ± 0.2% to 6.7 ± 0.2%. By contrast, the mesophyll tissue, which inversely related to that of mechanical tissue (Figure 5b), indicating a occupied the largest fraction (52.2 ± 0.9% to 64.3 ± 0.9%) of the trade‐off between assimilative and support tissues. cross‐sectional area, decreased with increasing needle length by 23% (Figures 2 and S1g). The decrease of mesophyll tissue cross‐sectional area with needle 3.2 | Variation in needle hydraulics, gas exchange, length was associated with a tendency for increasing mesophyll cell and N with needle length size (Figure 3a). In addition, the mesophyll thickness was greater for the two species with the shortest needles compared with the other The ratio of circle sector‐based estimate of circumference to mea- species except P. elliottii (maximum P = 0.003; Figure 3b). The sured circumference (Ccircle sector/C) of only the two P. taeda families combined effect of these responses was a decrease in total mesophyll was significantly different from 1 (one‐sample t test, P < 0.05). The surface area per leaf area (Smesophyll‐surf./Sneedle) with needle length deviation, however, was small (up to 4%; Table S2), and the regression (Figure 3c). Leaf morphological attributes (width, thickness, and cir- between circle sector‐based circumference and measured circumfer- cumference; Table 1) including needle cross‐sectional area (Figure 4 ence across species was not different from the 1:1 line (Figure S2). a) were insensitive to needle length, consistent with published data Thus, the circle sector seems to represent the needle cross section

(Figure 1b, Table S1). Leaf mass per area (LMAtotal‐surf.), which reflects of these species reasonably well, and using this simplified approxima- differences in both thickness and density among needles, was unre- tion was unlikely to affect the interpretation of the results. 2 lated to needle length (R = 0.40, P = 0.17). However, the fraction of Leaf hydraulic conductance (Kleaf) was positively correlated with mechanical or supporting tissue (epidermal tissue plus xylem) needle length (Figure 6a), increasing from 0.46 ± 0.13 to increased 35% from the shortest to the longest needles (Figure 4b), 2.39 ± 0.35 mmol m−2 s−1 MPa−1, or by 418% from the shortest to whereas both fresh and dry densities decreased 25% and 21%, respec- the longest needles. Moreover, one component of Kleaf, the xylem tively (Figure 4c). Both xylem tracheid diameter and hydraulic hydraulic conductance (Kxylem), increased with needle length across

FIGURE 2 Needle cross sections, leaf size, and the relationships between needle length and fractions of various needle tissues in five Pinus species (maximum P = 0.01). Symbols represent species mean values (n = 5) and error bars represent standard error 1696 WANG ET AL.

FIGURE 3 Variation in mesophyll cell size (a), mesophyll thickness (b), and mesophyll cells surface area per needle surface area

(Smesophyll‐surf./Sneedle; c) with needle length in five Pinus species. Symbols represent species means (n = 5), and error bars represent standard error. Solid and dotted lines represent regressions with P < 0.05 and P < 0.10, respectively. PV: Pinus virginiana; PEC: Pinus echinata; PT2 and PT1: Pinus taeda; PEL: Pinus elliottii; PP: Pinus palustris species by 420% (Figure 6b), with the variation in K explaining xylem FIGURE 4 Variation in needle mechanical, morphological, and 83% of that in Kleaf across species (Figure 6c). anatomical characteristics with needle length in five Pinus species. We found a strong relationship between the light‐saturated (a) Needle cross‐sectional area, (b) mechanical tissue fraction, (c) dry photosynthetic rate per unit needle surface area (Aarea) and stomatal (and fresh) density, and (d) tracheid diameter and hydraulic diameter. Symbols represent species means (n = 5), and error bars conductance (gs) within species (Figure S3a). gs and intercellular‐to‐ represent standard error. PV: Pinus virginiana; PEC: Pinus ambient (CO2) ratio (Ci/Ca) declined with increasing leaf‐to‐air water echinata; PT2 and PT1: Pinus taeda; PEL: Pinus elliottii; PP: Pinus VPD for most species (Figure S3b,c). Positive relation between A area palustris and gs was also observed among species (Figure 7a). However, it should be noted that, compared with hydraulic properties, the range of variation among species in all gas exchange‐based variables was WANG ET AL. 1697

in Narea with increasing needle length was attributable to a decrease in the fraction of mesophyll tissue (Figure 9). Summarizing, although

mesophyll tissue fraction and Narea decreased with increasing needle

length, Ci/Ca and Vc,max‐area were unresponsive, and gs and Aarea tended to increase.

4 | DISCUSSION

Our results demonstrate that needle anatomy varies with needle length among the five pine species studied (Figures 2 and S1). The anatomical differences reflected adjustments in the capacity for

mechanical support and leaf hydraulic conductance (Kleaf) as needles increased in length, suggesting coordination among leaf traits. With increasing needle length, the fraction of mechanical tissue increased,

whereas tissue density decreased (Figure 4). The increase of Kleaf was likely related to larger mean hydraulic diameter (Figure 4d) and shorter pathway for water movement through the mesophyll (Figure 3; Scoffoni et al., 2016). Changes in these trait values were, in turn, accompanied with similar (in direction) changes in gas

exchange characteristics, such as stomatal conductance (gs) and light‐

saturated photosynthetic rate per unit leaf area (Aarea; Figure 7).

4.1 | Needle length and traits related to mechanical support

FIGURE 5 Variation in needle anatomical traits with mechanical tissue fraction in five Pinus species. (a) Epidermal tissues fraction and Following our first hypothesis (H1a), we show that mechanical tissue xylem fraction, and (b) mesophyll tissue fraction. Symbols represent volume fraction increases with needle length (Figure 4b), consistent species means (n = 5), and error bars represent standard error. PV: with the increased requirements for support, as the bending moment Pinus virginiana; PEC: Pinus echinata; PT2 and PT1: Pinus taeda; PEL: operating on a needle increases with length (Gere & Timoshenko, Pinus elliottii; PP: Pinus palustris 1997; Niklas, 1992, 1999). Moreover, similar to the findings on volume fractions in three Mediterranean pines (Kuusk, Niinemets, & substantially smaller. P. palustris with the longest needles showed the Valladares, 2018a), we found a trade‐off between the relative −2 −1 −2 highest gs (110 ± 28 mmol m s ) and Aarea (7.2 ± 0.6 μmol m s fractions of support versus photosynthetic tissue (mesophyll) across −1 −2 −1 ), whereas P. echinata had the lowest gs (79 ± 17 mmol m s ) species (Figure 5b). Our results suggest that increasing structural −2 −1 and Aarea (6.1 ± 0.6 μmol m s ). Across all species, Ci/Ca showed stiffness comes at the cost of tissues involved in photosynthesis, in no trend with needle length (Figure 7b), whereas gs tended to increase both relative and absolute terms because the cross‐sectional area with needle length driving a similar trend in Aarea (Figure 7c). Conse- did not increase with needle length (Figure 4a). Our results add to quently, Aarea showed a tendency to increase with Kleaf (Figure 7d). the previous findings on positive relation between leaf size and invest-

Light‐saturated photosynthetic rate per unit mass (Amass) showed no ment to supporting tissues (Givnish, 1984; Niinemets, Portsmuth, & trend with needle length across species (Figure S4a). Tobias, 2006; Niinemets & Sack, 2006). They suggest that leaf size,

Although Aarea tended to increase with needle length across here determined primarily by needle length, strongly influences ana- species, maximum carboxylation capacity on area basis (Vc, max‐area) tomical traits and that redistribution from photosynthetic to mechan- and on mass basis (Vc, max‐mass) did not. Thus, the observed variation ical tissues as needles grow longer may be necessary to maintain in light‐saturated photosynthetic rate was unrelated to the maximum surface exposure to incoming light. rates of carboxylation. Furthermore, N content on needle surface area Among the supporting tissues, the xylem fraction (of the needle basis (Narea) decreased with needle length (Figure 8a) with an esti- cross section) was found most sensitive to changes in needle length. −2 mated maximum Narea of 1.02 ± 0.08 g m for P. echinata and a min- Increases in leaf size would inevitably require increased support and −2 imum of 0.69 ± 0.04 g m for P. palustris. Hence, Aarea was inversely transpiration requirements at the leaf scale. Our results suggest that, correlated with Narea (Figure 8b), and the variation in Vc,max‐area was although there may be a trade‐off between investments in light unrelated to Narea (Figure 8c). N content on mesophyll cell surface harvesting (i.e., interception of incoming radiation supported by area basis (Nmesophyll‐surf) was insensitive to needle length mechanical tissue), versus carbon fixation (photosynthesis tissue), the 2 (Figure S4b, R = 0.04, P = 0.70). Nitrogen concentration (Nmass) trade‐off is less costly for photosynthesis than it may appear for two showed no trend with needle length (Figure 8d) and did not explain reasons. First, investing more in xylem is an investment in both the the variation in either Amass or Vc, max‐mass (Figure 8e,f). The decrease mechanical tissue facilitating light capture (by reducing needle bending 1698 WANG ET AL.

FIGURE 6 Variation in needle hydraulic properties in five Pinus species. Leaf hydraulic conductance (Kleaf; a), xylem hydraulic conductance

(Kxylem) and outside‐xylem hydraulic conductance (Koutside‐xylem; b) as a function of needle length, and Kleaf as a function of Kxylem (c) and

Koutside‐xylem (d). Symbols represent species means (n = 5), and error bars represent standard error. Solid and dotted lines represent regressions with P < 0.05 and P < 0.10, respectively. PV: Pinus virginiana; PEC: Pinus echinata; PT2 and PT1: Pinus taeda; PEL: Pinus elliottii; PP: Pinus palustris and self‐shading) and the water transport system (by satisfying the reduced the rate of increase in mechanical support required to counter greater demand for hydraulic conductivity), thereby reducing stomatal the gravity effect on needle display. limitation to photosynthesis. Second, the properties of the tissues also changed with increasing needle length such that total needle volume density decreased (Figure 4c). As a result, the gravitational pull from its own weight per unit of length tended to decline (Figure S4c), reduc- 4.2 | Needle length and hydraulics ing the mechanical force resulting from the increasing load applied along the needle. Thus, the increased investment in mechanical sup- We found that Kleaf scaled with needle length across species (Figure 6 port is less than expected based on the change in length (Niklas, a), consistent with the increasing need for water transport (H1b). The 1992), and conversely, the decrease in photosynthetic tissue was not differences observed in maximum Kleaf among species were associated as drastic as it might have been without adjustments to the properties with differences in xylem hydraulic conductance (Kxylem; Figure 6c), in of the tissues. agreement with the pattern observed in other tree species (Domec The changes in needle density seem related to anatomical et al., 2016; Scoffoni et al., 2016). Xylem hydraulic conductance adjustments. Because epidermis occupies only a small volume fraction depends on both the tracheid lumen conductance (Klumen) and the and its density was shown to be low compared to other tissues pit membrane conductance (Kpits; Domec et al., 2016; Pittermann, (Poorter, Niinemets, Poorter, Wright, & Villar, 2009), we assumed that Sperry, Hacke, Wheeler, & Sikkema, 2006). Based on Hagen‐ the effect of variation of epidermis fraction on volume density was Poiseuille's law, Klumen scales with hydraulic diameter to the fourth negligible. Thus, the variation of density should be driven by changes power (Tyree & Ewers, 1991). Previous modelling studies have also in mesophyll and vascular tissue density and the shifts in their volume shown that Kxylem increases with more and larger xylem conduits fractions. Individual mesophyll cell size tended to increase with across species (Sack & Frole, 2006; Sommerville, Sack, & Ball, 2012). increasing needle length (Figure 3a), suggesting that mesophyll tissue In our study, the increase in hydraulic diameter with needle length density tended to decrease with needle length. Furthermore, tracheid (Figure 4d) suggested that Klumen increases with needle length. The ‐ diameter also increased with needle length (Figure 4d), implying that outside xylem hydraulic conductance (Koutside‐xylem) showed a similar the vascular tissue density decreased. Thus, although the vascular tis- trend (Figure 6b). We found that mesophyll cell size tended to sue density is higher than that of the mesophyll (Poorter et al., 2009) increase, yet the ratio of mesophyll cell surface area to needle surface and vascular tissue fraction increased with needle length in our study, area (Smesophyll‐surf./Sneedle) decreased with needle length (Figure 3), needle density decreased. The decrease in needle density with length likely reducing the path length of water movement from the xylem WANG ET AL. 1699

across the mesophyll tissue to the stomata in longer needles, poten-

tially explaining the increase in Koutside‐xylem (Brodribb et al., 2007).

It is noteworthy that the increase of Koutside‐xylem was not contin- uous with needle length, but our estimates for P. elliottii and P. palustris (two species with the longest needles) were much higher than the rest

of the species or, alternatively, the estimates of Koutside‐xylem of both P. taeda families were lower than expected based on needle length. It is possible that in order to satisfy the increased transpiration requirements of long needles with large surface area, in addition to decreasing the path length of water movement within mesophyll tis-

sue, other adjustments may be necessary to further increase Koutside‐

xylem. Based on previous studies, such strategies could include a com- bination of (a) increasing transfusion tissue fraction (Figure 2) to enhance radial water transport between the axial xylem and bundle sheath (Zhang, Rockwell, Wheeler, & Holbrook, 2014), and (b) improv- ing the aquaporin activity and/or density, which increase the perme- ability of membranes (Chaumont & Tyerman, 2014; Maurel, 1997). Alternatively, it is possible that the smaller mesophyll cell size of P. taeda (Figure 3a), perhaps associated with other unmeasured

changes, reduced Koutside‐xylem relative to that expected based on nee- dle length, despite having a thinner mesophyll (Figure 3b). Future research in needle anatomical traits could focus on the role of the

outside‐xylem structure in determining Koutside‐xylem.

4.3 | Needle length and gas exchange characteristics

Consistent with H1c, stomatal conductance (gs) tended to increase

with Kleaf, and needle length (Figure 7c,d), suggesting that among‐

species variation in needle anatomy and Kleaf may scale up to tree transpiration and leaf gas exchange (McCulloh et al., 2015; Sack &

Holbrook, 2006). When stomata are open to allow diffusion of CO2 into the leaf, water vapour is lost to the atmosphere, potentially dehydrating the leaf. The imbalance between the rate of evaporation and the rate of liquid water supply to the site of evaporation leads to substantial variation in leaf water potential, reflected in stomatal response designed to protect the xylem from cavitation (Oren et al., 1999; Sperry, Hacke, Oren, & Comstock, 2002). Thus, the capacity of to replace transpired water plays an important role in deter- mining the stomatal conductance and associated photosynthetic rates.

Indeed, because of that, Aarea and gs are highly correlated (Figure 7a; Damour, Simonneau, Cochard, & Urban, 2010; Wong, Cowan, &

Farquhar, 1979), and because gs is the gas‐phase equivalent of Kleaf,

it is not surprising that Aarea tended to increase with Kleaf (Figure 7d).

This finding is consistent with previous studies showing that Aarea of leaves is often limited by the ability of the leaf hydraulic system to

FIGURE 7 Relationships between (a) light‐saturated photosynthetic maintain leaves sufficiently hydrated for stomata to remain fully open rate on total needle surface area basis (Aarea) and stomatal (Brodribb, Holbrook, Zwieniecki, & Palma, 2005; Hao, Wheeler, conductance (gs), (b) intercellular to ambient CO2 concentration ratio Holbrook, & Goldstein, 2013). Therefore, increased Kleaf with needle (C /C ) and needle length, (c) g , A and needle length, and (d) g , A i a s area s area length appears to exert the greatest influence on Aarea. and leaf hydraulic conductance (Kleaf) in five Pinus species. Symbols The overwhelming control over Aarea by needle‐scale hydraulic represent species means (n = 10 for gs and Aarea and n = 5 for Kleaf), properties refutes our final hypothesis (H2) that across the five pine and error bars represent standard error. Solid and dotted lines represent regressions with P < 0.05 and P < 0.10, respectively. PV: species, Aarea is also reflecting the prevailing light conditions on the Pinus virginiana; PEC: Pinus echinata; PT2 and PT1: Pinus taeda; PEL: needle surfaces as affected by differences in within‐shoot mutual Pinus elliottii; PP: Pinus palustris shading across species (Figure 1a; Niinemets, Tobias, et al., 2006; 1700 WANG ET AL.

FIGURE 8 Variation in needle N content (total surface area; Narea) with needle length (a), and in area‐based light‐saturated photosynthetic rate

(Aarea) and maximum carboxylation capacity on total needle surface area basis (Vc,max‐area) with Narea (b, c). Variation in needle N concentration

(Nmass) with needle length (d), and in mass‐based light‐saturated photosynthetic rate (Amass), and maximum carboxylation capacity (Vc, max‐mass) with

Nmass (e, f). Symbols represent species means (n = 5), and error bars represent standard error. PV: Pinus virginiana; PEC: Pinus echinata; PT2 and PT1: Pinus taeda; PEL: Pinus elliottii; PP: Pinus palustris

FIGURE 9 Variation in needle N content (total surface area; Narea.) with (a) mesophyll tissue fraction, (b) mechanical tissue fraction, and (c) mesophyll cell surface area per needle surface area (Smesophyll‐surf./Sneedle) in five Pinus species. Symbols represent species means (n = 5), and error bars represent standard error. PV: Pinus virginiana; PEC: Pinus echinata; PT2 and PT1: Pinus taeda; PEL: Pinus elliottii; PP: Pinus palustris WANG ET AL. 1701

Stenberg et al., 1994; Thérézien et al., 2007). Such effect has been correlations of leaf functional traits in conifers might deviate from shown numerous times within species in response to growing‐light the universal relationships, which potentially indicate multiple drivers environment and within‐canopy light gradients, in both conifers and of leaf trait variation at different taxonomic scales (Anderegg et al., broadleaved species (e.g., Niinemets, Keenan, & Hallik, 2015; 2018; Osnas et al., 2018).

Niinemets, Lukjanova, Turnbull, & Sparrow, 2007; Oren, Schulze, As a final note, we wish to point out that, in this study, gs and A Matyssek, & Zimmermann, 1986; Stenberg et al., 2001), presumably were estimated at the leaf scale, although the actual consequences due to changes in leaf thickness, mesophyll fraction, and mesophyll‐ of various structural designs on carbon gain and water use must ulti- cell‐to‐needle‐surface‐area ratio (Smesophyll‐surf./Sneedle; Nobel et al., mately be evaluated at scales from shoots (the functional unit) to 1975). Here, however, differences among species in the interception crowns and canopies. An ongoing work is aimed at measuring and efficiency may be too small for the presumed effects on photosyn- scaling hydraulic traits to match the modelled mean light environment thetic capacity to clearly manifest. This is especially so considering and to assess whether these up‐scaled photosynthetic and hydraulic that the two hypothesized responses (light‐driven and hydraulics‐ capacities (or rates) are coordinated, thus matching mean stomatal driven) would produce similar effect on photosynthesis of increasing conductance with mean light availability. with needle length at the low‐mid portion of the range, with diverging effects noticeable only at the higher end. Summarizing, across species, ACKNOWLEDGEMENTS positive trends in Kleaf, gs, and Aarea with needle length conflicted with N.W. gratefully acknowledges the financial support from the China an inverse linear relation in Smesophyll‐surf./Sneedle (Figure 3c), suggesting Scholarship Council for the study abroad at Duke University. Financial that the hydraulic control over photosynthetic rate dominated over support for R.O. was provided by the Erkko Visiting Professor ‐ other leaf scale controls of photosynthetic capacity. Programme of the Jane and Aatos Erkko 375th Anniversary Fund, Indeed, in addition to inverse patterns of Aarea and Smesophyll‐surf./ through the University of Helsinki. This research was also partially ‐ ‐ Sneedle with needle length, both area based and mass based estimates supported by a grant from the National Science Foundation (NSF‐ of needle photosynthetic capacity across the pines were poorly IOS‐1754893). We thank Juan Chen and Yizheng Wang for their help related (or inversely related) to indicators of biochemical capacity. in the field and the NC State University Cooperative Tree Improve- Maximum carboxylation capacity (Vc, max), whether expressed on mass ment Program and the USDA Forest Service for access to the Longleaf or area basis, showed no trend with needle length and was unrelated Pine Regional Provenance/Progeny Trial in the Duke Forest. to variation in foliar N (Figure 8c,f). Variations in Amass were not reflecting those in foliar N concentration (Figure 8e). The decrease in ORCID N with increasing needle length (Figure 8a) generated an inverse area Na Wang https://orcid.org/0000-0002-3063-1706 relationship between A and N (Figure 8b), which further area area Sari Palmroth https://orcid.org/0000-0002-1290-4280 suggests that photosynthetic nitrogen use efficiency (A/N) increased Jean‐Christophe Domec https://orcid.org/0000-0003-0478-2559 with needle length. The tendency of A to increase with needle area Ram Oren https://orcid.org/0000-0002-5654-1733 length was mainly driven by a similar tendency in gs (Figure 7), although the decrease in N with needle length likely reflected the area REFERENCES redistribution of tissue between mesophyll and mechanical support Anderegg, L. D., Berner, L. T., Badgley, G., Sethi, M. L., Law, B. E., & (Figure 9), suggested by the insensitivity of Nmesophyll‐surf to needle HilleRisLambers, J. (2018). Within‐species patterns challenge our length (Figure S4b). 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Sommerville, K. E., Sack, L., & Ball, M. C. (2012). Hydraulic conductance of SUPPORTING INFORMATION Acacia phyllodes (foliage) is driven by primary nerve (vein) conductance and density. Plant, Cell and Environment, 35, 158–168. https://doi.org/ Additional supporting information may be found online in the 10.1111/j.1365‐3040.2011.02425.x Supporting Information section at the end of the article. Sperry, J., Hacke, U., Oren, R., & Comstock, J. (2002). Water deficits and ‐ hydraulic limits to leaf water supply. Plant, Cell and Environment, 25, Table S1: Needle length and needle cross sectional area from pub- 251–263. https://doi.org/10.1046/j.0016‐8025.2001.00799.x lished papers. 1704 WANG ET AL.

Table S2: The ratios of circle sector‐based estimate circumference and response of gs (b), and intercellular to ambient CO2 concentration

(Ccircle sector) to measured circumference (C), and of pie‐shaped esti- ratio (Ci/Ca) (c), to leaf‐to‐air water vapour pressure deficit in five Pinus mate circumference (Cpie) to measured circumference (C) in five Pinus species. Symbols represent individual needle samples (n = 10). Solid species (mean ± SE, n = 5). The difference between the ratios and 1 line represents relationship with P < 0.05, and dotted line represents were tested using one‐sample t‐test. relationship with P < 0.10. PV: Pinus virginiana, PEC: P. echinata, PT2 and PT1: P. taeda, PEL: P. elliottii, PP: P. palustris. FIGURE S1: Variation in mesophyll phloem fraction (a), xylem fraction (b), vascular bundle fraction (c), transfusion tissue fraction (d), central FIGURE S4: Variation in light‐saturated photosynthetic rate on mass cylinder fraction (e), epidermal tissue fraction (f) and mesophyll tissue basis (Amass) (a), needle N content on mesophyll cells surface area basis fraction (g) with needle length in five Pinus species. Symbols represent (Nmesophyll‐surf.) (b), and leaf mass per unit of needle length (LML) (c) species means (n = 5) and error bars represent standard error. PV: with needle length in five Pinus species. Symbols represent species

Pinus virginiana, PEC: P. echinata, PT2 and PT1: P. taeda, PEL: P. elliottii, means (n = 10 for Amass, and n = 5 for Nmesophyll‐surf. and LML) and error PP: P. palustris. bars represent standard error. PV: Pinus virginiana, PEC: P. echinata, PT2 and PT1: P. taeda, PEL: P. elliottii, PP: P. palustris. FIGURE S2: Correlations between circle sector‐based circumference, pie‐shaped‐based circumference and measured circumference with 95% confidence interval in five Pinus species. Symbols represent spe- How to cite this article: Wang N, Palmroth S, Maier CA, cies means (n = 5) and error bars represent standard error. The dotted Domec J‐C, Oren R. Anatomical changes with needle length line demonstrates the 1:1 line. PV: Pinus virginiana, PEC: P. echinata, are correlated with leaf structural and physiological traits PT2 and PT1: P. taeda, PEL: P. elliottii, PP: P. palustris. across five Pinus species. Plant Cell Environ. 2019;42: FIGURE S3: Light‐saturated photosynthetic rate on total needle sur- 1690–1704. https://doi.org/10.1111/pce.13516 face area basis (Aarea) as a function of stomatal conductance (gs) (a), 1 Table S1 Needle length and needle cross-sectional area from published papers.

Species Name Needle Cross-sectional Area Reference Length (mm2) (cm) Picea mariana 1.3 0.68 Weng & Jackson, 2000 Abies balsamea 1.33 0.76 Piene & Percy, 1984 Picea omorika 1.36 0.83 Nikolić, Bojović & Marin, 2015 Picea sitchensis 1.6 0.86 Weng & Jackson, 2000 Picea rubens 1.65 0.44 Weng & Jackson, 2000 Picea glauca 1.7 1.10 Weng & Jackson, 2000 Picea breweriana 1.8 0.95 Weng & Jackson, 2000 Picea martinezii 1.95 0.45 Weng & Jackson, 2000 Picea engelmannii 2.05 1.78 Weng & Jackson, 2000 Picea pungens 2.05 1.93 Weng & Jackson, 2000 2.2 1.13 Weng & Jackson, 2000 Picea mexicana 2.8 1.46 Weng & Jackson, 2000 Pinus uliginosa 4.75 0.62 Boratyńska et al., 2008 Pinus echinata 5 0.46 Thérézien, Palmroth, Brady & Oren, 2007 Pinus rigida 5 0.64 Zwieniecki et al., 2006 Pinus sylvestris 5.07 0.56 Boratyńska, Jasińska & Ciepłuch, 2008 Pinus sylvestris 5.07 1.08 Lin, Jach & Ceulemans, 2001 Pinus radiata 7.5 0.35 Conroy, Barlow & Bevege, 1986 Pinus canariensis 8.1 0.26 Grill, Tausz, Pöllinger, Jiménez & Morales, 2004 Pinus halepensis 10.74 0.46 Esteban, Martín, de Palacios, Fernández & López, 2010 Pinus halepensis 10.93 0.56 Esteban, Martín, de Palacios, Fernández & López, 2010 Pinus halepensis 11.92 0.49 Esteban, Martín, de Palacios, Fernández & López, 2010 Pinus taeda 12.3 0.49 Niinemets, Ellsworth, Lukjanova & Tobias, 2002 Pinus taeda 15 0.65 Thérézien et al., 2007 Pinus ponderosa 20 1.04 Zwieniecki et al., 2006 Pinus palustris 29.1 0.79 Niinemets, Ellsworth, Lukjanova & Tobias, 2002 Pinus palustris 30 0.87 Thérézien et al., 2007

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25 Thérézien M., Palmroth S., Brady R. & Oren R. (2007) Estimation of light interception properties of

26 conifer shoots by an improved photographic method and a 3D model of shoot structure. Tree

27 Physiology, 27, 1375-1387.

28 Weng C. & Jackson S.T. (2000) Species differentiation of North American spruce (Picea) based on

29 morphological and anatomical characteristics of needles. Canadian Journal of Botany, 78, 1367-

30 1383. 31 Zwieniecki M.A., Stone H.A., Leigh A., Boyce C.K. & Holbrook N.M. (2006) Hydraulic design of pine

32 needles: one-dimensional optimization for single-vein leaves. Plant, Cell and Environment, 29,

33 803-809.

34 35 Table S2 The ratios of circle sector-based estimate circumference (Ccircle sector) to measured circumference

36 (C), and of pie-shaped estimate circumference (Cpie) to measured circumference (C) in five Pinus species

37 (mean ± SE, n=5). The difference between the ratios and 1 were tested using one-sample t-test.

species Ccircle sector/ C Ccircle sector/ C Cpie/ C Cpie/ C P value P value P. virginiana (PV) 1.017 ± 0.003 0.09 0.977 ± 0.006 0.03

P. echinata (PEC) 0.997 ± 0.006 0.66 0.968 ± 0.002 0.0005

P. taeda (PT2) 1.025 ± 0.004 0.006 0.963 ± 0.002 0.0009 (PT1) 1.036 ± 0.002 0.0006 0.965 ± 0.001 0.00002 P. elliottii (PEL) 0.998 ± 0.011 0.85 0.961 ± 0.004 0.001

P. palustris (PP) 1.010 ± 0.004 0.08 0.976 ± 0.002 0.0005 38 Figures

39 FIGURE S1. Variation in mesophyll phloem fraction (a), xylem fraction (b), vascular bundle fraction (c),

40 transfusion tissue fraction (d), central cylinder fraction (e), epidermal tissue fraction (f) and mesophyll

41 tissue fraction (g) with needle length in five Pinus species. Symbols represent species means (n = 5) and

42 error bars represent standard error. PV: Pinus virginiana, PEC: P. echinata, PT2 and PT1: P. taeda, PEL:

43 P. elliottii, PP: P. palustris.

44 FIGURE S2. Correlations between circle sector-based circumference, pie-shaped-based circumference

45 and measured circumference with 95% confidence interval in five Pinus species. Symbols represent

46 species means (n = 5) and error bars represent standard error. The dotted line demonstrates the 1:1 line.

47 PV: Pinus virginiana, PEC: P. echinata, PT2 and PT1: P. taeda, PEL: P. elliottii, PP: P. palustris.

48 FIGURE S3. Light-saturated photosynthetic rate on total needle surface area basis (Aarea) as a function of

49 stomatal conductance (gs) (a), and response of gs (b), and intercellular to ambient CO2 concentration ratio

50 (Ci/Ca ) (c), to leaf-to-air water vapour pressure deficit (VPD) in five Pinus species. Symbols represent

51 individual needle samples (n = 10). Solid line represents relationship with P<0.05, and dotted line

52 represents relationship with P<0.10. PV: Pinus virginiana, PEC: P. echinata, PT2 and PT1: P. taeda,

53 PEL: P. elliottii, PP: P. palustris.

54 FIGURE S4. Variation in light-saturated photosynthetic rate on mass basis (Amass) (a), needle N content

55 on mesophyll cells surface area basis (Nmesophyll-surf.) (b), and leaf mass per unit of needle length (LML) (c)

56 with needle length in five Pinus species. Symbols represent species means (n = 10 for Amass, and n = 5 for

57 Nmesophyll-surf. and LML) and error bars represent standard error. PV: Pinus virginiana, PEC: P. echinata,

58 PT2 and PT1: P. taeda, PEL: P. elliottii, PP: P. palustris. 59

60 Figure S1

Figure S2

Figure S3

Figure S4